Measurement of the blood coagulation time is carried out for the screening of the presence or absence of any abnormality in the blood coagulation system, or for the measurement of the activity of individual blood coagulation factors, by measuring the time period starting from the time point at which a reagent for blood coagulation time measurement including an activating agent for blood coagulation factors (hereinafter, may be simply referred to as activating agent) and/or Ca2+ and the like is added to a specimen blood or a specimen blood mixture, to the time point at which detectable fibrin clots are formed (blood coagulation time; hereinafter, may also be simply referred to as coagulation time. Also, formation of fibrin clots may also be simply referred to as coagulation). Typical examples of blood coagulation tests include prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. Hereinafter, in the present specification, blood coagulation factors may be simply referred to as coagulation factors.
PT is the time taken from the addition of a mixed liquid of tissue thromboplastin and Ca2+ to a test plasma, to the occurrence of coagulation, and this is intended to comprehensively examine the coagulation activities of factor VII, factor X, factor V, prothrombin, fibrinogen, and the like that are associated with the extrinsic pathway of coagulation. Furthermore, APTT is the time taken from the addition of a sufficient amount of phospholipids and an activating agent (kaolin, anhydrous silicic acid, ellagic acid, or the like) and an appropriate amount of Ca2+ to a test plasma, to the occurrence of coagulation, and this is intended to comprehensively examine the coagulation activity of factor XII, factor XI, prekallikrein, high molecular weight kininogen, factor IX, factor VIII, factor X, factor V, prothrombin, fibrinogen and the like, which are associated with the intrinsic pathway of coagulation. In general, what is referred to as abnormality in these blood coagulation tests refers to the prolongation of the coagulation time. Abnormality in the blood coagulation system reflects the signs or results of the tendency to hemorrhage or the tendency to thrombosis (tendency to blood coagulation) in the body.
Regarding the causes for the prolongation of the coagulation time, the following can be considered: 1) deficiency or a decrease in blood coagulation factors, 2) the presence of an antibody to a blood component that constitutes the blood coagulation system, 3) the presence of an antibody to a component in the reagent for blood coagulation time measurement, 4) the presence of an antibody to a complex of a blood component that constitutes the blood coagulation system and a component in the reagent for blood coagulation time measurement, and 5) administration of a drug that inhibits the blood coagulation reaction.
However, simply performing the measurement of the blood coagulation time does not enable discriminating whether the cause of the prolongation of the coagulation time is, for example, a decrease in the blood coagulation activity due to simple deficiency of coagulation factors, or a decrease in the blood coagulation activity due to inhibition of the coagulation reaction by an antibody (inhibitor) to a component that constitutes the blood coagulation system or a component in the reagent for blood coagulation time measurement. On the other hand, since the therapeutic policy varies with the difference in the relevant cause of prolongation, discrimination of the cause of prolongation is important. Thus, there has been a blood coagulation correction test (hereinafter, also may be referred to as “blending test” or “mixing test”) in which for the purpose of determination of the cause of prolongation, normal plasma is added to a test plasma, and the extent to which the blood coagulation time of the test plasma is corrected (normalized) is plotted into a graph to determine the cause (Non-Patent Document 1).
Conventionally, the mixing test has been carried out, for example, in the manner described below.
Samples are prepared by adding and mixing normal plasma to a test plasma such that the mixing proportions of the normal plasma are 0%, 20%, 50%, 80% and 100%, and the APTT is measured. The results are plotted into a graph (horizontal axis: proportion of normal plasma incorporated or the proportion (%) of the test plasma, vertical axis: coagulation time (seconds)), and the cause of prolongation of the coagulation time is visually discriminated and determined from the shape of the graph. For example, when the test plasma is coagulation factor-deficient, the addition of a small amount of normal plasma (20% in FIG. 1(A)) significantly shortens the coagulation time so that the coagulation time approaches close to the value obtainable when 100% normal plasma is measured. Therefore, the graph shows a downward convex curve below a straight line (dotted line) that connects the points corresponding to 100% test plasma and 100% normal plasma (FIG. 1(A)).
When a coagulation factor inhibitor is present in a test plasma, the relevant coagulation factor inhibitor inactivates coagulation factors in the added normal plasma, even though the proportion of addition of normal plasma is increased. Therefore, the extent of improvement in the coagulation time due to the addition of normal plasma is low, and a curve that is convex upward is shown (FIG. 1(B)).
As a coagulation factor inhibitor which affects the sensitivity of the reagent for blood coagulation time measurement, lupus anticoagulant (hereinafter, LA) is known. LA is defined as an immunoglobulin which inhibits a phospholipid-dependent coagulation reaction in vitro without inhibiting the activity of individual coagulation factors, and is not a single antibody. Since the presence of phospholipids is essential to the coagulation reaction, usually, many of the reagents for blood coagulation time measurement are rich in phospholipids. LA reacts with phospholipids in the reagents, thereby consuming these phospholipids, and consequently inhibits the coagulation reaction to prolong the coagulation time. Therefore, the results of coagulation tests such as PT and APTT are often found to be abnormal. However, since LA varies in reaction intensities depending on the type of phospholipids (origin, phospholipid composition, and the like), it is known that different results of determination on LA positivity/negativity are obtained depending on the reagent for blood coagulation time measurement to be used.